ABSTRACT:The peripheral conversion of testosterone to estradiol by aromatase is the primary source of endogenous estrogen in postmenopausal women. Studies indicating that placental aromatase is able to metabolize methadone to its primary metabolite, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidin (EDDP), led us to test the hypothesis that methadone is able to act as an inhibitor of aromatase. Using recombinant human CYP19, we examined the ability of methadone to bring about either reversible or mechanism-based inhibition of the conversion of testosterone to estradiol. To test for reversible inhibition, racemic methadone or its metabolite EDDP or 2-ethyl-5-methyl-3, 3-diphenylpyrroline (EMDP) was incubated for 30 min with testosterone at the K m (4 M). To test for mechanismbased inhibition, microsomal preincubations were performed for up to 30 min using racemic methadone (1-1000 M), R-or Smethadone (0.5-500 M), or EDDP or EMDP (10 and 100 M) followed by incubation with testosterone at a V max concentration (50 M). Racemic methadone, EDDP, and EMDP did not act as competitive inhibitors of CYP19. Preincubation of methadone, EDDP, or EMDP with CYP19 resulted in time-and concentrationdependent inhibition, indicating a mechanism-based reaction that destroys CYP19 activity. The K I and k inact values for racemic methadone were calculated to be 40.6 ؎ 2.8 M and 0.061 ؎ 0.001 min ؊1, respectively. No stereoselectivity was observed. Methadone is metabolized by CYP19 and may act as a potent inhibitor of CYP19 in vivo. These findings may contribute to variability in methadone clearance, to drug-drug interactions, and to side effects observed in individual patients.